Small volume bioreactors with substantially constant working volumes and associated systems and methods

ABSTRACT

Control of volume in bioreactors and associated systems is generally described. Feeding and/or sampling strategies can be employed, in some embodiments, such that the working volume within the bioreactor remains substantially constant.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofInternational Application Number PCT/US2014/052252, filed Aug. 22, 2014,entitled “Small Volume Bioreactors with Substantially Constant WorkingVolumes and Associated Systems and Methods” which claims priority toU.S. Provisional Patent Application Ser. No. 61/869,116, filed Aug. 23,2013, and entitled “Small Volume Bioreactors with Substantially ConstantWorking Volumes and Associated Systems and Methods,” each of which isincorporated herein by reference in its entirety for all purposes.International Application Number PCT/US2014/052252 also claims priorityto European Patent Application Number EP 14306161, filed Jul. 17, 2014,and entitled “Small Volume Bioreactors With Substantially ConstantWorking Volumes and Associated Systems and Methods,” which isincorporated herein by reference in its entirety for all purposes.

TECHNICAL FIELD

Control of volume in small volume bioreactors and associated systems isgenerally described.

SUMMARY

Small volume bioreactors with substantially constant working volumes,and associated systems and methods, are generally described. In certainembodiments, feeding and/or sampling strategies can be employed suchthat the working volume within the bioreactor remains substantiallyconstant. The bioreactors may be operated in a fed-batch mode ofoperation, in some embodiments. The subject matter of the presentinvention involves, in some cases, interrelated products, alternativesolutions to a particular problem, and/or a plurality of different usesof one or more systems and/or articles.

Certain embodiments relate to a method of operating a bioreactor. Themethod comprises, in some embodiments, performing, within thebioreactor, a biochemical reaction in which at least one eukaryotic cellis grown within a liquid medium having a volume of less than about 50milliliters; adding a first amount of liquid to the liquid medium in thebioreactor during the biochemical reaction; and removing a second amountof liquid from the liquid medium in the bioreactor during thebiochemical reaction. In some such embodiments, during at least about80% of the time over which the biochemical reaction is performedincluding during the adding and removing steps, the total volume ofliquid within the bioreactor does not fluctuate by more than about 20%from an average of the volume of liquid within the bioreactor.

In certain embodiments, the method comprises performing, within thebioreactor, a biochemical reaction within a liquid medium having avolume of less than about 50 milliliters; adding a first amount ofliquid to the liquid medium in the bioreactor during the biochemicalreaction; and removing a second amount of liquid from the liquid mediumin the bioreactor during the biochemical reaction. In some suchembodiments, during at least about 80% of the time over which thebiochemical reaction is performed including during the adding andremoving steps, the total volume of liquid within the bioreactor doesnot fluctuate by more than about 20% from an average of the volume ofliquid within the bioreactor, and the osmolarity of the liquid mediumwithin the bioreactor is maintained within a range of from about 200osmoles per kilogram of the liquid medium to about 600 osmoles perkilogram of the liquid medium.

According to some embodiments, the method comprises performing, withinthe bioreactor, a biochemical reaction in which at least one eukaryoticcell is grown within a liquid medium having a volume of less than about50 milliliters; adding a first amount of liquid to the liquid medium inthe bioreactor during a first period of time over which the biochemicalreaction is performed; and removing a second amount of liquid having avolume that is within 10% of a volume of the first amount of liquid fromthe liquid medium in the bioreactor during a second period of time overwhich the biochemical reaction is performed that does not overlap withthe first period of time; and repeating the adding a removing steps atleast one time. In some such embodiments, the adding step and theremoving step are performed such that, between the adding step and theremoving step, substantially no liquid is removed from the bioreactorvia a non-evaporative pathway, and substantially no liquid is added tothe bioreactor.

The method comprises, in certain embodiments, performing, within thebioreactor, a biochemical reaction within a liquid medium having avolume of less than about 50 milliliters; adding a first amount ofliquid to the liquid medium in the bioreactor during a first period oftime over which the biochemical reaction is performed; removing a secondamount of liquid having a volume that is within 10% of a volume of thefirst amount of liquid from the liquid medium in the bioreactor during asecond period of time over which the biochemical reaction is performedthat does not overlap with the first period of time; and repeating theadding a removing steps at least one time. In some such embodiments, theadding step and the removing step are performed such that, between theadding step and the removing step, substantially no liquid is removedfrom the bioreactor via a non-evaporative pathway, and substantially noliquid is added to the bioreactor. In some such embodiments, during atleast about 80% of the time over which the biochemical reaction isperformed, the osmolarity of the liquid medium within the bioreactor ismaintained within a range of from about 200 osmoles per kilogram of theliquid medium to about 600 osmoles per kilogram of the liquid medium.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 is an exemplary cross-sectional side view schematic illustrationof a bioreactor, according to one set of embodiments;

FIG. 2 is, according to certain embodiments, a cross-sectional side viewschematic illustration of an exemplary reactor system;

FIGS. 3A-3C are cross-sectional side view schematic illustrations of areactor chamber and a mode of operating the same, according to someembodiments;

FIG. 4 is a bottom-view cross sectional schematic illustration of areactor system including a plurality of reactor chambers arranged inseries, according to one set of embodiments;

FIG. 5 is an exemplary top-view schematic illustration of amicro-bioreactor, according to one set of embodiments;

FIG. 6 is, according to certain embodiments, a top-view schematicillustration of the connectivity between the bioreactor and a gasmanifold;

FIG. 7 is a schematic illustration of an exemplary gas manifold,according to some embodiments;

FIG. 8 is a table outlining the operation of a plurality of valves in anexemplary bioreactor, according to one set of embodiments;

FIG. 9 is a photograph of an exemplary bioreactor system, according tocertain embodiments; and

FIG. 10 is, according to some embodiments, a side-view cross sectionalschematic illustration of an exemplary bioreactor, according to someembodiments.

DETAILED DESCRIPTION

Control of volume in bioreactors and associated systems is generallydescribed. Feeding and/or sampling strategies can be employed, in someembodiments, such that the working volume within the bioreactor remainssubstantially constant.

In certain embodiments, the bioreactors described herein are configuredto contain a relatively small volume of liquid (e.g., less than 50milliliters). For many small volume bioreactors, even small changes involume can lead to large deviations in the process conditions, such asgas transfer rate, mixing rate, and the like. According to certainembodiments, strategies are employed such that the volume of the liquidwithin the bioreactor is kept constant even after feeding and/orsampling have occurred. For example, in certain instances, the volume ofliquid within the bioreactor is maintained such that the volume does notdeviate from the average volume by more than 20%. This can be achieved,for example, by adjusting the volume of liquid removed from and/or addedto the bioreactor such that the volume of liquid within the bioreactoris maintained within a relatively narrow range of volumes. For example,in certain embodiments, if the volume of the liquid within thebioreactor increases beyond a certain level, the sampling volume isincreased so as to keep the volume within the desired range. In someembodiments, if the volume of the liquid within the bioreactor decreasesbelow a certain level, the volume of liquid fed to the bioreactor can beincreased to maintain a substantially constant volume of liquid withinthe bioreactor.

The bioreactors described herein can be configured to perform a varietyof suitable biochemical reactions. In some embodiments, the bioreactorcan be configured to grow at least one biological cell. The cells withinthe bioreactor can be suspended in a liquid medium, such as any commoncell growth medium known to those of ordinary skill in the art. Certainembodiments involve performing, within the bioreactor, a biochemicalreaction in which at least one eukaryotic cell is grown within a liquidmedium having a volume of less than about 50 milliliters.

In some embodiments, the osmolarity of the liquid medium within thebioreactor can be maintained within a desirable range. For example, incertain embodiments, the osmolarity of the liquid medium within thebioreactor can be maintained within a range of from about 200 osmolesper kilogram of the liquid medium to about 600 osmoles per kilogram ofthe liquid medium. Maintaining the osmolarity within this range can beuseful for growing eukaryotic cells, which generally require differentsalinity conditions than prokaryotic cells for proper cell growth.

As noted above, in some embodiments, the volume of the liquid within thebioreactor is maintained within a desirable range. This can be helpful,according to certain embodiments, in controlling conditions within thebioreactor during the biochemical reaction (e.g., during cell growth).Certain embodiments comprise adding a first amount of liquid (e.g.,containing at least one biochemical reactant, such as a cell growthmedium) to the liquid medium in the bioreactor during the biochemicalreaction and removing a second amount of liquid (e.g., containing atleast one biochemical reaction product, such as a biological cell) fromthe liquid medium in the bioreactor during the biochemical reaction. Thefirst amount of liquid can be added over a first period of time overwhich the biochemical reaction is performed. The second amount of liquidcan be removed during second period of time over which the biochemicalreaction is performed. In some embodiments, the second period of timedoes not substantially overlap with the first period of time. The addingand removing steps can be repeated at least one time during operation ofthe bioreactor.

In some such embodiments, the amounts of liquid added and/or removed canbe selected such that during at least about 80% of the time over whichthe biochemical reaction is performed including during the adding andremoving steps, the total volume of liquid within the bioreactor doesnot fluctuate by more than about 20% from an average of the volume ofliquid within the bioreactor. In certain embodiments, the second amountof liquid that is removed from the bioreactor has a volume that iswithin 10% of the volume of the first amount of liquid that is added tothe bioreactor. In some embodiments, the second volume of liquid isadded during a second period of time over which the biochemical reactionis performed that does not overlap with the first period of time (i.e.,the first period of time during which the first amount of liquid isadded to the liquid medium). In certain embodiments, the adding step andthe removing step are performed such that, between the adding step andthe removing step, substantially no liquid is removed from thebioreactor via a non-evaporative pathway, and substantially no liquid isadded to the bioreactor.

FIG. 1 is a schematic cross-sectional illustration of bioreactor 100,according to one set of embodiments. In FIG. 1, bioreactor 100 comprisesbioreactor chamber 102. Bioreactor 100 can be configured to perform abiochemical reaction. In some embodiments, the bioreactor can beconfigured to grow at least one biological cell. In some suchembodiments, operation of the bioreactor comprises growing at least oneeukaryotic cell. For example, operating the bioreactor may comprise, insome embodiments, growing at least one animal cell, such as a mammaliancell. In some embodiments, operating the bioreactor comprises growing atleast one Chinese hamster ovary (CHO) cell. In certain embodiments, thebioreactor is configured to grow an invertebrate cell (e.g., a cell froma fruit fly), a fish cell (e.g., a zebrafish cell), an amphibian cell(e.g., a frog cell), a reptile cell, a bird cell, a primate cell, abovine cell, a horse cell, a porcine cell, a goat cell, a dog cell, acat cell, or a cell from a rodent such as a rat or a mouse. In someembodiments, the bioreactor can be configured to grow at least one humancell. If the cell the bioreactor is configured to grow is from amulticellular organism, the cell may be from any part of the organism.For instance, if the cell is from an animal, the cell may be a cardiaccell, a fibroblast, a keratinocyte, a heptaocyte, a chondracyte, aneural cell, a osteocyte, a muscle cell, a blood cell, an endothelialcell, an immune cell (e.g., a T-cell, a B-cell, a macrophage, aneutrophil, a basophil, a mast cell, an eosinophil), a stem cell, etc.In some cases, the cell may be a genetically engineered cell. While cellgrowth, and in particular eukaryotic cell growth, is primarily describedherein, it should be understood that other types of biochemicalreactions could also be performed using bioreactor 100. For example, insome embodiments, the bioreactor can be used to produce a protein.

In certain embodiments, the bioreactor contains a liquid. For example,referring back to the exemplary embodiment of FIG. 1, bioreactor chamber102 contains a liquid 104. The liquid within the bioreactor maycomprise, in certain embodiments, a cell growth medium. In embodimentsin which one or more cell growth media are employed, any suitable typeof medium can be used, including any common cell-growth mediumcontaining essential amino acids and/or cofactors known to those ofordinary skill in the art. In embodiments in which the bioreactor isconfigured to perform other types of biochemical reactions, the liquidwithin the bioreactor may contain any other suitable precursor forperforming the biochemical reaction.

The liquid medium within the bioreactor may occupy a relatively smallvolume, in certain embodiments. For example, in some embodiments, theliquid medium has a volume of less than about 50 milliliters, less thanabout 10 milliliters, or less than about 5 milliliters (and/or incertain embodiments, as little as 1 milliliter, 0.1 milliliters, orless). The use of small volume bioreactors can be desirable, forexample, when one wishes to perform parallel analysis of a large numberof bioreactor conditions while using relatively small amounts of inputmaterial (e.g., cells, growth medium, etc.). However, the use of suchsmall volumes can present challenges. For example, accurate control ofreaction conditions can be relatively difficult to achieve in such smallvolume reactors. As noted above, certain aspects of the presentinvention relate to feeding and/or sampling techniques that allow forthe effective operation of bioreactors having very small volumes.

Certain embodiments relate to adding liquid to and/or removing liquidfrom the bioreactor such that the volume of the liquid within thebioreactor is maintained substantially constant. This can be achieved,according to certain embodiments, by adding liquid to and/or removingliquid from the bioreactor in controlled amounts such that the volume ofliquid within the bioreactor does not exceed a maximum allowable valueand/or does not drop below a minimum acceptable value. In certainembodiments, the bioreactor may be operated in a fed-batch mode ofoperation.

In some embodiments, operation of the bioreactor comprises adding afirst amount of liquid to the liquid medium in the bioreactor (e.g.,during the biochemical reaction). The first amount of liquid can beadded to the liquid medium in the bioreactor during a first period oftime over which the biochemical reaction is performed within thebioreactor. In certain embodiments, the liquid that is added to thebioreactor can be at least a portion of a makeup stream, which can beused to maintain the volume of liquid within the bioreactor within anacceptable range of volumes. In some embodiments, the liquid that isadded to the liquid medium in the bioreactor comprises at least onebiochemical reactant. For example, the liquid that is added to theliquid medium in the bioreactor can comprise an essential amino acid, acofactor, and/or any other suitable reactant useful for performing abiochemical reaction. In certain embodiments, the composition of theliquid that is added to the bioreactor can be substantially the same asthe composition of the liquid within the bioreactor. For example, theliquid that is added to the bioreactor may contain the same or similargrowth medium as is contained within the bioreactor during operation. Byadding liquid having a composition that is substantially the same as thecomposition of the liquid within the bioreactor, one or more propertiesof the liquid within the bioreactor (e.g., pH, osmolarity, nutrientconcentration, etc.) can be maintained substantially constant. In otherembodiments, the composition of the liquid that is added to thebioreactor may be different than the composition of the liquid mediumcontained within the bioreactor during operation. In some embodiments,the liquid that is added to the liquid medium in the bioreactor is notsubstantially pure water. For example, the liquid that is added to theliquid medium in the bioreactor may be an aqueous composition containingwater and at least one other component. In some embodiments, the liquidthat is added to the liquid medium in the bioreactor is a non-aqueouscomposition.

Operating the bioreactor may involve, in certain embodiments, removing asecond amount of liquid from the liquid medium in the bioreactor (e.g.,during the biochemical reaction). The second amount of liquid can beremoved from the bioreactor during a second period of time over whichthe biochemical reaction is performed within the bioreactor. In certainembodiments, the second amount of liquid that is removed from thebioreactor comprises a product of a biochemical reaction. For example,the second amount of liquid that is removed from the bioreactor cancomprise, in some embodiments, a biological cell. In some embodiments,the second amount of liquid that is removed from the bioreactor cancomprise a protein. In certain embodiments, the second amount of liquidthat is removed from the liquid medium in the bioreactor is notsubstantially pure water. For example, the liquid that is removed fromthe liquid medium in the bioreactor can be, in some embodiments, anaqueous composition containing water and at least one other component.In some embodiments, the liquid that is removed from the liquid mediumin the bioreactor is a non-aqueous composition.

In certain embodiments, the liquid that is removed from the bioreactormay be removed as part of a sampling procedure. For example, in certainembodiments, operating the bioreactor comprises determining at least oneproperty (e.g., pH, osmolarity, component concentration, and/ortemperature) of the liquid that is removed from the bioreactor. In someembodiments, operating the bioreactor comprises altering at least oneproperty of the bioreactor (e.g., pH and/or temperature) and/or of aliquid input stream (e.g., pH, temperature, flow rate, and/orcomposition) at least partially in response to the determination of theproperty of the liquid that is removed from the bioreactor.

Liquid can be added to and/or removed from the bioreactor via anysuitable pathway. In some embodiments, adding the first amount of liquidto the liquid medium within the bioreactor comprises transporting thefirst amount of liquid into the bioreactor via a liquid inlet. Forexample, referring to FIG. 1, the first amount of liquid (and/or,subsequent amounts of liquid) can be added to bioreactor 100 via liquidinlet 114. The liquid inlet can comprise, in certain embodiments, achannel, such as a microfluidic channel. For example, referring to FIG.1, liquid inlet 114 corresponds to a microfluidic liquid inlet channelfluidically connected to reactor chamber 102.

In some embodiments, removing the second amount of liquid from theliquid medium in the bioreactor comprises transporting the second amountof liquid out of the bioreactor via a liquid outlet. For example,referring to FIG. 1, the second amount of liquid (and/or, subsequentamounts of liquid) can be removed from bioreactor 100 via liquid outlet115. The liquid outlet can comprise, in certain embodiments, a channel,such as a microfluidic channel. For example, referring to FIG. 1, liquidoutlet 115 corresponds to a microfluidic liquid outlet channelfluidically connected to reactor chamber 102.

Transporting liquid into and/or out of the bioreactor can be achievedusing any suitable method. For example, in some embodiments, a pressuregradient can be established by applying a positive pressure to the inletof a channel using, for example, a pump, via gravity, or by any othersuitable method. In some embodiments, pressure gradients within achannel can be established by applying a negative pressure to an outletof a channel, for example, via attachment of a vacuum pump to an outlet,withdrawal of air from a syringe attached to an outlet, or by any othersuitable method. Fluid transport can also be achieved using peristalticpumping configurations, including those described elsewhere herein.

In certain embodiments, the adding and removing steps can be performedsuch that the volume of the liquid medium within the bioreactor remainssubstantially constant during the time over which the biochemicalreaction (e.g., cell growth) is performed. For example, in someembodiments, during at least about 80% (or at least about 90%, at leastabout 95%, or at least about 99%, and/or, in certain embodiments, up to100%) of the time over which the biochemical reaction is performedincluding during the adding and removing steps, the total volume ofliquid within the bioreactor does not fluctuate by more than about 20%(or more than about 10%, or more than about 5%) from the average of thevolume of liquid within the bioreactor. One of ordinary skill in the artwould be capable of determining the average of the volume of the liquidwithin the bioreactor during a biochemical reaction by, for example,monitoring the volume of the liquid within the bioreactor during thetime over which the biochemical reaction is performed and calculating athe average of the volume as a time-averaged value.

According to certain embodiments, the volume of the first amount ofliquid that is added to the bioreactor and the volume of the secondamount of liquid that is removed from the bioreactor are relativelyclose. In some such embodiments, removing amounts of liquid that areclose in volume to the amounts of added liquid can ensure that theliquid level in the bioreactor is maintained within a desired range ofvolumes during operation of the bioreactor. In some embodiments,operating the bioreactor comprises adding a first amount of liquid tothe bioreactor and removing a second amount of liquid from thebioreactor, wherein the second amount of liquid has a volume that iswithin 10% of (or within 5% of, within 1% of, and/or, in certainembodiments, substantially the same as) the volume of the first amountof liquid.

In some embodiments, the steps of adding liquid to and removing liquidfrom the bioreactor are performed as temporally separate steps. Incertain embodiments, the step of adding the first amount of liquid isperformed over a first period of time, the step of removing the secondamount of liquid is performed over a second period of time, and thefirst and second periods of time do not substantially overlap with eachother. For example, in some embodiments, the step of adding the firstamount of liquid may be performed first, and after the adding step hasbeen completed, the step of removing the second amount of liquid may beperformed. In some embodiments, the step of removing the second amountof liquid may be performed first, and after the removing step has beencompleted, the step of adding the first amount of liquid may beperformed. It should be understood, however, that separate adding andremoving steps are not required in all embodiments, and that in someinstances, the adding and removing steps may at least partially (or maysubstantially completely) overlap.

In certain embodiments, the adding step and/or the removing step areperformed such that, between the adding step and the removing step,substantially no liquid is removed from the bioreactor via anon-evaporative pathway. For example, referring back to FIG. 1, in someembodiments, during operation of bioreactor 100, substantially no liquidis removed from the bioreactor via liquid outlet 115 between the addingstep and the removing step. In some embodiments, the adding step and theremoving step are performed such that, between the adding step and theremoval step, substantially no liquid is removed from the bioreactor(e.g., via an evaporative pathway, such as through a vapor-permeablemembrane, or via any other pathway).

In some embodiments, the adding step and/or the removing step areperformed such that, between the adding step and the removing step,substantially no liquid is added to the bioreactor. For example,referring back to FIG. 1, in some embodiments, during operation ofbioreactor 100, substantially no liquid is added to the bioreactor vialiquid inlet 114 between the adding step and the removing step.

The steps of adding liquid and removing liquid can be repeated,according to certain embodiments, any number of times. In someembodiments, the steps of adding liquid and removing liquid can berepeated at least one time, at least two times, at least five times, atleast ten times, or at least 100 times (and/or, in certain embodiments,up to 1000 times, up to 10,000 times, or more) during the biochemicalreaction.

In certain embodiments, the osmolarity of the liquid medium within thebioreactor is maintained within a desired range of values. Maintainingthe osmolarity within a desired range can be beneficial, for example,when the bioreactor is used to grow cells that are sensitive to theconcentration of salt within the liquid growth medium. For example,eukaryotic cells can be, in some instances, sensitive to the osmolarityof the liquid medium in which they are grown. In some such instances, ifthe osmolarity of the liquid growth medium is too high, water may flowout of the cell, which can damage the cell membrane and render itmetabolically inactive. In addition, in some such instances, if theosmolarity of the liquid growth medium is too low, water may flow intothe cell, which may cause the cell to burst. Eukaryotic cells can beparticularly sensitive to the osmolarity of the liquid growth medium. Inaddition, eukaryotic cells generally require osmotic conditions that aredifferent from (and, in many cases, harder to achieve) those required byprokaryotic cells.

In some embodiments, the osmolarity of the liquid medium within thebioreactor is maintained within a range of from about 200 osmoles perkilogram of the liquid medium to about 600 osmoles per kilogram of theliquid medium (and/or, in certain embodiments, from about 300 osmolesper kilogram of the liquid medium to about 500 osmoles per kilogram ofthe liquid medium). In some embodiments, during at least about 80% (orat least about 90%, at least about 95%, or at least about 99%, and/or,in certain embodiments, up to 100%) of the time over which thebiochemical reaction is performed including during the adding andremoving steps, the osmolarity of the liquid medium within thebioreactor is maintained within a range of from about 200 osmoles perkilogram of the liquid medium to about 600 osmoles per kilogram of theliquid medium (or, in certain embodiments, from about 300 osmoles perkilogram of the liquid medium to about 500 osmoles per kilogram of theliquid medium). In some embodiments, maintaining the osmolarity of theliquid growth medium within a desired range can be accomplished, forexample, by adding liquid to the bioreactor that has an osmolarity thatis within 10% of, within 5% of, within 1% of, or substantially the sameas the osmolarity of the liquid within the bioreactor. For example, onecan add a liquid growth medium to the bioreactor having a similar saltconcentration as that of the liquid growth medium already contained inthe bioreactor.

While FIG. 1 illustrates one type of bioreactor configuration that maybe employed in association with certain of the embodiments describedherein, other types of bioreactors could also be used. One such exampleis illustrated in FIG. 2. In FIG. 2, bioreactor 200 comprises bioreactorchamber 202. Bioreactor chamber 202 can comprise a gaseous headspace206. Gaseous headspace 206 can be positioned above liquid growth medium204 in bioreactor chamber 202. In certain embodiments, gaseous headspace206 and liquid growth medium 204 can be in direct contact. In suchsystems, interface 208 in FIG. 2 can correspond to a gas-liquidinterface. In other embodiments, gaseous headspace 206 and liquid growthmedium 204 are separated by a moveable wall. For example, interface 208can correspond to a flexible membrane. In embodiments in which suchflexible membranes are employed, the membrane can be permeable to atleast one gas. For example, the flexible membrane can be, in certainembodiments, permeable to oxygen and/or carbon dioxide.

In certain embodiments, reactor chamber 202 comprises a first inlet 210connecting a source 212 of gas to gaseous headspace 206. Source 212 canbe any suitable source, such as a gas tank. The gas within gaseousheadspace may be used to actuate the movement of interface 208 and/or todeliver gas to and/or remove gas from liquid medium 204. Source 212 cancontain any suitable gas such as carbon dioxide, oxygen (which can beused to aerate liquid growth medium 204), and/or an inert gas (such ashelium or argon, which might be used to actuate interface 208 to producemixing within liquid growth medium 204, as described in more detailelsewhere). Optionally, reactor chamber 202 can comprise outlet 211,which can be used to transport gas out of gaseous headspace 206.Reactors employing arrangements similar to those described with respectto FIG. 2 are described, for example, in U.S. Patent Publication No.2013/0084622 by Ram et al., filed Sep. 30, 2011, and entitled “Deviceand Method for Continuous Cell Culture and Other Reactions” and U.S.Patent Application Publication No. 2005/0106045 by Lee, filed Nov. 18,2003, and entitled “Peristaltic Mixing and Oxygenation System,” each ofwhich is incorporated herein by reference in its entirety for allpurposes.

FIGS. 3A-3C are cross-sectional schematic illustrations outlining howfluid can be transported by deflecting a moveable wall into and out of aliquid sub-chamber of a reactor chamber. In FIGS. 3A-3C, reactor system300 comprises reactor chamber 302. In certain embodiments, reactorchamber 302 in FIGS. 3A-3C corresponds to reactor chamber 202 in FIG. 2.Reactor chamber 302 can comprise a liquid sub-chamber 303. Liquidsub-chamber 303 can be configured to contain a liquid growth mediumincluding at least one biological cell. Reactor chamber 302 cancomprise, in certain embodiments, gas sub-chamber 306. Gas sub-chamber306 can be configured to contain a gaseous headspace above the liquidgrowth medium within liquid sub-chamber 303.

Reactor chamber 302 can also comprise a moveable wall 308, which canseparate liquid sub-chamber 303 from gas sub-chamber 306. Moveable wall308 can comprise, for example, a flexible membrane. In certainembodiments, the moveable wall is formed of a medium that is permeableto at least one gas (i.e., a gas-permeable medium). In certainembodiments, for example, moveable wall can be permeable to oxygen gasand/or carbon dioxide gas. In such embodiments in which moveable wall308 is permeable to a gas (e.g., oxygen and/or carbon dioxide), the gaswithin gas sub-chamber 306 can be transported to liquid sub-chamber 303,or vice versa. Such transport can be useful, for example, to transportoxygen gas into a liquid medium within liquid sub-chamber 303 and/orcontrol pH by transporting carbon dioxide into or out of liquidsub-chamber 303.

Reactor system 300 can comprise, in certain embodiments, a gas inletconduit 304, which can be configured to transport gas into gassub-chamber 306. Gas inlet conduit 304 in FIGS. 3A-3C can correspond tothe gas inlet conduit 210 illustrated in FIG. 2, in certain embodiments.The gas that is transported into gas sub-chamber 306 can originate from,for example, gas source 316. Any suitable source of gas can be used asgas source 316, such as gas cylinders. In certain embodiments, gassource 316 is a source of oxygen and/or carbon dioxide.

In some embodiments, reactor system 300 comprises gas outlet conduit 312configured to transport gas out of gas sub-chamber 306. Gas outletconduit 312 in FIGS. 3A-3C can correspond to the gas outlet conduit 211illustrated in FIG. 2, in certain embodiments. In some embodiments,reactor system 300 comprises gas bypass conduit 310 connecting gas inletconduit 304 to gas outlet conduit 312. Gas bypass conduit 310 can beconfigured such that it is external to reactor chamber 302, in certainembodiments. Reactor system 300 can also comprise, in certainembodiments, a liquid inlet conduit 311 and a liquid outlet conduit 314.

In certain embodiments, moveable wall 308 can be actuated such that thevolumes of liquid sub-chamber 303 and gas sub-chamber 306 are modified.For example, certain embodiments involve transporting a gas from gassource 316 through gas inlet conduit 304 to gas sub-chamber 306 todeform moveable wall 308. Deformation of moveable wall 308 can beachieved, for example, by configuring reactor 300 such that gassub-chamber 306 is pressurized when gas is transported into gassub-chamber 306. Such pressurization can be achieved, for example, byrestricting the flow of gas out of gas outlet conduit 312 (e.g., usingvalves or other appropriate flow restriction mechanisms) while gas isbeing supplied to gas sub-chamber 306.

In certain embodiments, deforming moveable wall 308 can result in liquidbeing at least partially evacuated from liquid sub-chamber 303. Forexample, in FIG. 3B, moveable wall 308 has been deformed such thatsubstantially all of the liquid within liquid sub-chamber 303 has beenevacuated from reactor chamber 302. Such operation can be used totransport the liquid within liquid sub-chamber 303 to other liquidsub-chambers in other reactors, as illustrated, for example, in FIG. 4,described in more detail below.

In certain embodiments, after at least a portion of the liquid withinliquid sub-chamber 303 has been removed from liquid sub-chamber 303, thesupply of the gas to gas sub-chamber 306 can be reduced such thatmoveable wall 308 returns toward its original position (e.g., theposition illustrated in FIG. 3A). In certain embodiments, moveable wall308 will be deflected such that at least a portion of the gas within gassub-chamber 306 is removed from the gas sub-chamber. Such gas might beremoved, for example, if liquid enters liquid sub-chamber 303 fromliquid inlet conduit 311, for example, from another upstream reactor, asdescribed in more detail below.

Certain embodiments include the step of supplying gas from gas source316 to gas sub-chamber 306 at least a second time to deform moveablewall 308 such that liquid is at least partially removed from liquidsub-chamber 303. When such gas introduction steps are performedrepeatedly, moveable wall 308 can act as part of a pumping mechanism,transporting liquid into and out of liquid sub-chamber 303. Suchoperation is described in detail in U.S. Patent Publication No.2013/0084622 by Ram et al, filed Sep. 30, 2011, and entitled “Device andMethod for Continuous Cell Culture and Other Reactions,” which isincorporated herein by reference in its entirety for all purposes.

In certain embodiments in which gas is transported into gas sub-chamber306 multiple times, gas can be transporting from the gas source throughgas bypass conduit 310. Transporting gas through gas bypass conduit 310can be performed to remove liquid from gas inlet conduit 304 withouttransporting the liquid to gas sub-chamber 306. For example, in certainembodiments, a first valve between gas bypass conduit 310 and gas inlet305 can be closed and a second valve between gas bypass conduit 310 andgas outlet 307 can be closed (and any valves within gas bypass conduit310 can be opened) such that, when gas is transported through gas inletconduit 304, the gas is re-routed through gas bypass conduit 310, andsubsequently out gas outlet conduit 312. Such operation can serve toflush any unwanted condensed liquid out of the gas inlet conduit, whichcan improve the performance of the gas supply methods describedelsewhere herein.

In some embodiments, multiple sets of reactor chambers can be arranged(e.g., in series) such that fluidic mixing is achieved along one or morefluidic pathways. FIG. 4 is a bottom view, cross-sectional schematicdiagram illustrating the liquid flow paths that can be used to establishmixing between multiple reactor chambers 102A-C connected in series, asdescribed in U.S. Patent Publication No. 2013/0084622 by Ram et al,filed Sep. 30, 2011, and entitled “Device and Method for Continuous CellCulture and Other Reactions,” which is incorporated herein by referencein its entirety for all purposes.

In FIG. 4, reactor system 400 includes a first fluidic pathway indicatedby arrows 410. The first fluidic pathway can include a first reactorchamber 102A, a second reactor chamber 102B, and a third reactor chamber102C. Reactor system 400 also includes conduits 421, 422, and 423, whichcan correspond to liquid inlet and/or liquid outlet conduits for reactorchambers 102A-C. For example, in FIG. 4, conduit 421 is a liquid inletconduit for reactor chamber 102B and a liquid outlet conduit for reactorchamber 102A; conduit 422 is a liquid inlet conduit for reactor chamber102C and a liquid outlet conduit for reactor chamber 102B; and conduit423 is a liquid inlet conduit for reactor chamber 102A and a liquidoutlet conduit for reactor chamber 102C. Of course, the flow of liquidcan also be reversed such that conduits 421, 422, and 423 assumeopposite roles with respect to each of reactor chambers 102A-C.

Reactor system 400 can also include a liquid input conduit 450 and aliquid output conduit 451, which can be used to transport liquid intoand out of the liquid sub-chambers within reactor chambers 102A, 102B,and 102C. Valve 452 may be located in liquid input conduit 450, andvalve 453 may be located in liquid output conduit 451 to inhibit orprevent to the flow of liquid out of the mixing system during operation.

In certain embodiments, the moveable walls of reactor chambers 102A-Ccan be actuated to transport liquid along fluidic pathway 410 (and/oralong a fluidic pathway in a direction opposite pathway 410). This canbe achieved, for example, by sequentially actuating the moveable wallswithin reactor chambers 102A-C such that liquid is transported in acontrolled direction. In some embodiments, each of reactor chambers102A-C can be configured such that they are each able to assume a closedposition wherein moveable wall 308 is strained such that the volume ofthe liquid sub-chamber is reduced, for example, as illustrated in FIG.3B. Peristaltic mixing can be achieved, for example, by actuatingreactor chambers 102A-C such that their operating states alternatebetween open (FIG. 3A or FIG. 3C) and closed (FIG. 3B) configurations.In some embodiments, three patterns may be employed to achieveperistaltic pumping: a first pattern in which the liquid sub-chamber ofreactor chamber 102A is closed and the liquid sub-chambers withinreactor chambers 102B and 102C are open; a second pattern in which theliquid sub-chamber of reactor chamber 102B is closed and the liquidsub-chambers within reactor chambers 102A and 102C are open; and a thirdpattern in which the liquid sub-chamber of reactor chamber 102C isclosed and the liquid sub-chambers within reactor chambers 102A and 102Bare open. By transitioning among these three patterns (e.g., changingfrom the first pattern to the second pattern, from the second pattern tothe third pattern, and from the third pattern to the first pattern,etc.) liquid can be transported among reactor chambers 102A-C in aclockwise direction (as illustrated in FIG. 4). Of course, byre-arranging the order in which the patterns occur (e.g., by changingfrom the first pattern to the third pattern, from the third pattern tothe second pattern, and from the second pattern to the first pattern,etc.), liquid can be transported in the counter-clockwise direction aswell.

The bioreactors described herein may be manufactured using a variety ofsuitable techniques. In some embodiments, the bioreactors are fabricatedusing standard microfabrication techniques. Such techniques may involvevarious film deposition processes (such as spin coating, atomic layerdeposition, sputtering, thermal evaporation, electroplating, electrolessplating, and chemical vapor deposition), laser fabrication processes,photolithographic techniques, etching methods including wet chemical orplasma processes, and the like. In some embodiments, the bioreactors canbe fabricated using micromachining techniques. In certain embodiments,the bioreactors can be fabricated using molding techniques.

The systems described herein may be microfluidic, in some embodiments,although the invention is not limited to microfluidic systems and mayrelate to other types of fluidic systems. “Microfluidic,” as usedherein, refers to a device, apparatus or system including at least onefluid channel having a cross-sectional dimension of less than about 1mm. A “microfluidic channel,” as used herein, is a channel meeting thesecriteria. The “cross-sectional dimension” (e.g., a diameter) of thechannel is measured perpendicular to the direction of fluid flow. Insome embodiments, the devices described herein include at least onechannel having a maximum cross-sectional dimension of less than about500 micrometers, less than 200 micrometers, less than about 100micrometers, less than 50 micrometers, or less than about 25micrometers.

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

EXAMPLE 1

This example describes the operation of a bioreactor in which the liquidvolume within the reactor is maintained within a desired range whileliquid is added to and removed from the bioreactor.

Bench top bioreactors are the standards for scale down models ofindustrial bioreactors at a scale of 1000-10,000 times smaller thanindustrial bioreactors. Since volume and surface area scale differentlywith length, the physical and chemical environment experienced by thecells even in bench top bioreactors that are geometrically identical toindustrial bioreactors will be different. The physical and chemicalenvironment of the cells can strongly affect the cells' physiology andproductivity and hence should be maintained constant or within thelimits of critical values during scaling. First, the gas transfer rateof O₂ and CO₂ should be sufficiently high so that the dissolved oxygenlevel remains above the oxygen uptake rate of the cells and waste gaslike carbon dioxide are efficiently removed. Secondly, the maximum shearrate experienced by the cells should remain the same or below thecritical value that affects productivity during the scaling. This can beespecially important for mammalian cells like CHO due to their shearsensitivity. The circulation time is also an important parameter sinceit affects the frequency at which the cells experience high shear. Therepeated deformation of the endoplasmic reticulum has been reported toaffect protein glycosylation. Bioreactors with different chamber volumeswill have very different circulation time before the cells circulateback to the tip of the impeller and hence, some bench top bioreactorsare equipped with a circulation line that allows the physicalenvironment of the cells to mimic the circulation time seen in largeindustrial scale bioreactors. On the other hand, the mixing rate of themicro-bioreactor must be sufficiently fast and uniform so that there isno region in the culture where the cell is nutrient starved or have alarge concentration gradient. When designing scale down models ofbioreactors, the energy dissipation rate should be maintainedsubstantially constant so that the transfer of internal energy to thecell remains substantially constant.

Micro-bioreactors can be instrumented with online sensors like pH,dissolved oxygen (DO), dissolved carbon dioxide (DCO₂) and opticaldensity (OD) sensors. However, in order to fully characterize thecondition of the cell culture, offline sampling to monitor otherimportant culture parameters is generally desirable. Offline samplingfor cell viability measurements can be used to ensure that the cellviability remains high during the culture, since CHO cells are veryfragile. It would be desirable if cell viability could be measured inreal-time as an online sensor in the micro-bioreactor. For fed-batchcultures, where glucose is fed to the cells in the middle of theculture, osmolarity is generally an important parameter and cangenerally only be measured offline since it typically involves freezingthe sample to determine the freezing point. A high osmolarity canrepress cell metabolism and cause cell shrinkage. Next, to monitor cellhealth and productivity, it is desirable to measure (by conventionalmethods) the concentration of metabolites and product titer in theculture medium. These are generally measured offline since thesemeasurements involve the addition of reagents. In some instances, onlinesensors need to be recalibrated to account for any drifts during theculture, and hence, such measurements might need to be performed offlineusing a blood gas analyzer as a standard for comparison. Furthermore,end point measurements may be needed to ensure that the final productshave the right glycosylation, are not fragmented and have the rightpeptide groups and function. Generally, the volume of the bioreactorwill need to be sufficiently large to allow one to conduct these offlinesamples (and also for the end point protein titer and quality analysis)in order for these micro-bioreactors to function as well as bench topbioreactors.

Exemplary sample volume requirements for various commercially availableunits are summarized in Table 1.

TABLE 1 Offline sampling volume requirements for various commercialanalyzers. Required Sample No Instrument Volume Dilution Volume CellViability Measurements (5 Samples) 1 Hemacytometer 25 μL 1:1-1:10 2.5-25μL 2 Cedex HiRes Analyzer 300 μL  1:10 30 μL (Innovatis) 3 Vi-CELL 500μL  1:10 50 μL (Beckman Coulter) 4 Countess 10 μL 1:1-1:10 1-10 μL(Invitrogen) Blood Gas Analyzer (2 Samples) 1 Cobas b 221 50 μL 1:1 50μL (Roche) 2 Ciba Corning 840 45 μL 1:1 45 μL (Corning) Osmometer (5Samples) 1 Osmomat Auto 50 μL 1:1 50 μL (Gonotec) 2 5010 Osmette III 10μL 1:1 10 μL (PSi) 3 Model 20G Osmometer 20 μL 1:1 20 μL (AdvancedInstruments) Metabolites and Protein Titer (5 Samples) 1 RX Daytona 150μL  1:2 75 μL (Randox) 2 YSI 2700 Select 100 μL  1:2 50 μL (metabolitesonly) (YSI) 3 Octet QK (titer only) 100 μL  1:2 50 μL (Fortebio)

Since the samples for these instruments are typically taken from shakeflasks and large scale bioreactors, the recommended sample volumes forthese instruments can be rather large. For micro-bioreactors, dilutionsof the samples may be necessary to make up for the large volumerequired, since microbioreactors tend to have small working volumes. Forcell viability measurements, hemacytometer measurements or manualcounting under the optical microscope requires sample volume simplybecause only a small number of cells are counted, typically around 500cells per hemacytometer. Statistically, counting 1000 cells would meanthat the measured viability would lie within ±5% of the actual viabilityvalue of the population for 95% of the samples. If the hemacytometercounting were performed twice per measurement, an accuracy of ±5% couldbe achieved. However, to obtain a better accuracy, automated cellcounting methods, for example the Cedex HiRes, Vi-CELL and CountessAnalyzers, are generally used. The Cedex HiRes and Vi-CELL requires300-500 microliters of sample volume and allow the user to select thenumber of images they want counted from the sample. The larger thenumber of images counted, the smaller the error, but the imageprocessing will be time-consuming. Dilutions of up to 10 times arecommon when measuring samples with high cell density (e.g., about 10⁸cells/mL). The measurable cell densities are between 10⁴ and 10⁹cells/mL and hence, even at an incubation density of 2×10⁵ cells/mL, adilution of 10 times will still be within the measurement range. Sincemost users do not utilize all the images, a part of the sample will bediscarded without being counted. This is one reason why not allautomated cell counting machines require such a large volume. Countess,for example, requires only a 10 microliter sample volume and hence canbe used without requiring any dilution except for high cell densitycultures.

Other measurements that may be taken include offline pH, dissolvedoxygen (DO), and/or dissolved carbon dioxide (DCO₂) measurements, whichcan be performed using a blood gas analyzer. Since dissolved gas levelscan change when the sample is removed from the environment of the growthchamber of the bioreactor, this measurement should generally beperformed as fast as possible to prevent any degassing. Hence, thesamples for the blood gas analyzer cannot generally be diluted. Therecommended sample volumes for two commercial blood gas analyzers areshown in Table 1. The samples for offline osmolarity measurements usinga freezing point osmometer also generally cannot be diluted becauseosmolarity is not a linear function of concentration for most biologicalfluids. A freezing point osmometer operates by measuring the depressionof the freezing temperature due a change in chemical potential from thepresence of solutes in the solution. The sample size can be controlledby the size of the cooling chamber and temperature probe. From Table 1,one can see a wide range in recommended sample volumes for the freezingpoint osmometer. Another offline measurement that can be performed isthe measurement of the concentration of metabolites and product titer.The RX Daytona and YSI 2700 listed in Table 1 utilizes a pipette to drawout a fixed volume of samples to mix with different reagents that teststhe different components in the sample. The RX Daytona can measureconcentrations of glucose, glutamine, glutamate, lactate, ammonia andimmunoglobulin G (IgG) requiring only 57 microliters of sample volumefor the reagents. However, since the machine dips an automated pipetteinto a tube to draw out the required volume, the sample volume requiredalso depends on the depth of the pipette in the tube. It is believedthat the minimum sample volume at the operating height for the automatedpipette is 150 microliters. The sample can be diluted 2 fold to reducethe sample volume needed, and it is believed that any further dilutionwould result in the glutamine concentration dropping below themeasurement range for CHO media supplied with glutamate since glutaminewill only be synthesized by the cells as needed. For the YSI 2700analyzer, which measures the concentration of glucose, glutamate,glutamine and lactate, the pipetted volume is 25 microliters but theminimum sample volume needed for the operation of the machine is 100microliters. The YSI Analyzer is generally supplemented with the OctetQK for product titer measurements, requiring a sample volume of 100microliters. The recommended dilutions and final sample volume for eachmeasurement are listed in Table 1 together with the total number ofoffline samples needed for each parameter per 14 day CHO cell culture.From Table 1, it is estimated that the total volume removed for offlinesampling is approximately 650-1000 microliters, depending on whichinstruments are used.

For the end point analysis, Table 2 shows the protein weight needed forthe different downstream analysis of protein titer and quality.

TABLE 2 Downstream processing sampling volume requirements. VolumeVolume Minimum (700 (500 No Measurement Weight mg/L) mg/L) 1 SEC (SizeFragmentation) 20 μg 30 μL 60 μL 2 SDS - PAGE (Electrophoretic  4 μg 10μL 20 μL Fractionation) 3 Protein A HPLC (Purification) 20 μg 30 μL 60μL 4 HPAEC - PAD 200 μg  300 μL  600 μL  (Glycosylation) 5 WCX(Separation) 20 μg 30 μL 60 μL Total 264 μg  400 μL  800 μL 

The sample volume needed for downstream analysis are shown in Table 2for a product titer of 700 mg/L and 350 mg/L. The total volume neededfor the end point analysis is between 400-800 microliters. In order forthe micro-bioreactor to provide sufficient sample volume for offline anddownstream analysis, the working volume of the micro-bioreactor shouldgenerally be 2 mL or higher.

Maintaining high cell viability in the CHO cell population is animportant first step before any experiments on CHO cells can beperformed, be it in a micro-scale environment or a large scaleenvironment. In large scale environments, offline sampling is oftenperformed to monitor CHO cell population viability via a cell countingmethod either optically with exclusion dyes or electrically using aCoulter counter. Since micro-bioreactors typically have cell suspensionvolumes between 10 nL to 1 mL, offline cell counting methods, typicallyrequiring 20 L of sample each time, would significantly decrease thecell culture volume over a the culture period (typically 10 days). Formicro-bioreactors, an online method is preferred for cell viabilitymonitoring due to the small volume of the growth chamber. On the otherhand, the higher data density of online sensors would be an addedadvantage for research based studies in microenvironments. Since thesensors will be in contact with the cell suspension, there are verystringent requirements on the types of sensors that can be utilized foronline cell physiology monitoring. First, the online sensing methodshould be able to perform its measurement without a affecting cellviability, productivity, and physiological state. With live cells, theconditions of the media will also change over time due to cellmetabolism, hence a good online sensor must also be able to workreliably even under changing media conditions. Additionally, the sensorshould also be sterile and non-toxic during the entire duration of theexperiment and be compatible with common sterilization methods withoutcompromising the sensor's physical or chemical conditions. Dielectricspectroscopy (DS) for online cell viability monitoring can beparticularly useful, in certain cases, because it is label-free,scalable to micro-scale systems and compatible with most sterilizationmethods.

A new reactor design, referred to in this example as the ResistiveEvaporation Compensated Actuator (RECA) micro-bioreactor, which isillustrated in FIG. 5, has been developed for culturing cells, includingCHO cells. The reactor includes 5 reservoirs for injections, includingone containing sterile water for evaporation compensation. The otherfour reservoirs can be used for Sodium Bicarbonate (NaHCO₃) baseinjections, feed, and other necessary supplements. Injection can beperformed by a peristaltic pump actuated through the PDMS membranesequentially pushing a plug of fluid into the growth chamber. In thisexample, the growth chamber has a volume of 2 milliliters. Uniformmixing can be obtained by pushing fluids through small channelsconnecting the three growth chambers, each having a volume of 1milliliter. There is also a 10 microliter reservoir for sampling locatedafter the growth chamber. The sampling can be performed via peristalticpumping of 10 microliter plugs. Besides the connection to the growthchamber, the sample reservoir is also connected via a channel to thesterile water line and a clean air line. Air can be injected through thesample reservoir to eject any remaining sample into the samplingcontainer (e.g. an Eppendorf tube), and water can be injected after thatto clean the sample reservoir and remove any cell culture or cellsremaining. Clean air can then be sent through the reservoir to dry thechambers so that there is no water left to dilute the next sample. Thisprocess can be repeated after each sampling step.

The connections from the RECA micro-bioreactor to the gas manifold areshown in FIG. 6. All reservoir input valves can share the same gas linesince it is unnecessary to individually control each input valve. Thereservoir pressure can be set to be 1.5 psi (1.03×10⁵ Pa), which islower than that of the mixing pressure of 3 psi (2.06×10⁵ Pa). Thereservoir pressure can be used to ensure that the input to theperistaltic pumps sees the same pressure and is unaffected by externalhydrostatic pressure to ensure consistent pumping volume. The output ofthe reservoir, i.e. the injection valves, can be individually controlledby separate gas lines because these are the valves that determine whichfeed lines are being injected into the growth chamber. Next are the gaslines that control the peristaltic pumps. The mixers can have a separateinput and output line in order to allow flushing of water condensationon the mixer lines, since the air coming into the mixer can behumidified to reduce evaporation of the growth culture. The growthchambers of the micro-bioreactor have large surface to volume ratios andhence, the evaporation rates are generally larger than that for largerbioreactors. Moreover, all three mixer gas lines can be designed to havethe same resistance, to ensure an even mixing rate in the 3 growthchambers. The mixer gas lines can be made wider than the rest of thelines because the air is humidified, and any condensation might clog thelines if the resistance is too high. The last air lines control thevalves to the sampling port. The sampling port consists of a 10microliter sample reservoir and valves to control sampling and automatedcleaning of the sampling port. The holes in the top left corner can besealed with a polycarbonate cover and taped with double sided tape. Theair lines can be connected through a group of 20 barbs located on theleft bottom corner of the chip to the gas manifold.

A gas manifold can be used to connect the solenoid valves to the airlines of the micro-bioreactor. The design of the gas manifold is shownin FIG. 7. The manifold in this example has 3 layers. The barbconnectors to the micro-bioreactor are situated in the center of the toplayer of the manifold. The middle layer routes the output of thesolenoid valves to the barb connectors that connects the manifold to themicro-bioreactor. The bottom layer routes the main air lines to theinputs of the solenoid valves. FIG. 8 lists all the valves with theirnumbers as shown in FIG. 7 and the gas connections for easierreferencing. In the table, NO stands for Normally Open and NC stands forNormally Closed. The selection of which gas lines is normally open ornormally closed can be selected to be the most common state of thevalve, so that more often than not, the valve is inactive, to saveenergy consumption. In particular, Valve 10 (Pump 2) can be set to “off”normally while all the rest of the valves are set to “on” normally.There are also 4 gas mixer solenoid valves besides the solenoid valvesneeded for mixing and valving on the micro-bioreactor. Control of carbondioxide (CO₂) gas concentration vs. nitrogen (N₂) gas can be achieved bychanging the duty cycle of Gas Mix 3 solenoid valve. Oxygen (O₂) gasconcentration can be controlled via Gas Mix 2 via the same strategy.Then the two outputs can be mixed together in a 50-50 duty cycle usingGas Mix 1. Gas Mix 4 is available for use if any extra valving isneeded.

The complete setup is shown in FIG. 9. A laptop can be used to control aField-programmable Gate Array (FPGA) board, which can control thesolenoid boards, the heater board, and photo-detector board. Air linescan be connected to a pressure regulator before being connected to thegas manifold. From the gas manifold, the valve lines can be connecteddirectly to the micro-bioreactor. The mixer in lines are connected firstthrough an air resistance line, followed by a 45° C. local humidifierbefore reaching the micro-bioreactor. The mixer output lines from themicro-bioreactor are connected to the water trap, then to the airresistance lines and then only to the gas manifold.

Offline sampling of the bioreactor, if not compensated for, could causethe working volume of the bioreactor to be irregular throughout theculture. In addition, for fed-batch cultures where extra feed isinjected on certain days into the microbioreactor, in some cases thevolume of the liquid within the bioreactor could exceed the designedworking volume. The day to day volume variation expected for a batch andfed-batch 2 mL working volume CHO culture is summarized in Table 3.

TABLE 3 Day to day working volume of the RECA bioreactor for variousoperating schemes. All volumes are shown in microliters. 2 mL Batch 2 mLFed-Batch Normal Normal Over Sample Day Samp. Total Add Samp. Total AddSamp. Over Total 0 0 2000 0 0 2000 0 0 0 2000 1 0 2000 200 0 2200 200 0200 2000 2 0 2000 0 0 2200 0 0 0 2000 3 155 1845 220 155 2265 200 155 452000 4 0 1845 0 0 2265 0 0 0 2000 5 0 1845 227 0 2492 200 0 200 2000 6155 1690 0 155 2337 0 155 0 1845 7 50 1640 234 50 2520 185 50 0 1980 8 01640 0 0 2520 0 0 0 1980 9 155 1485 252 155 2617 198 155 23 2000 10 01485 0 0 2617 0 0 0 2000 11 0 1485 0 0 2617 0 0 0 2000 12 80 1405 0 802537 0 80 0 1920 13 75 1330 0 75 2462 0 75 0 1845 14 205 1125 0 205 22570 205 0 1640

If the volume in the mixer exceeds the designed maximum working volumeof 2 mL, the mixing will be incomplete and there will be dead zones inthe mixer as illustrated in FIG. 10. The dead zones will generally arisebecause the fluid will be stationary below the maximum deflection of themembrane. This can be a problem, especially for fed-batch cultures,since the volume of the micro-bioreactor is expected to exceed 2 mLthroughout the entire culture after Day 0.

One way to address the volume variations within the bioreactor is tooversample for the fed-batch culture. An exemplary oversampling strategyis shown in the last column of Table 3. Table 3 includes the expectedday to day working volumes (in microliters) of the RECA bioreactor forbatch culture, fed-batch culture, and fed-batch culture withoversampling to prevent over-filling of the bioreactor. The fed-batchprotocol is assumed to include a feed that leads to a 10% increase involume in days 1, 3, 5, 7, and 9. If the bioreactor is oversampled tomaintain a maximum volume of 2 mL, liquid can be added every day exceptDays 6-8 and Days 12-14. This is an additional advantage of theoversampling strategy. On days in which closed loop evaporationcompensation cannot be performed, injections of fluid can be made byadding an amount of fluid that corresponds to the amount of fluid lostfrom the bioreactor via evaporation.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,and/or methods, if such features, systems, articles, materials, and/ormethods are not mutually inconsistent, is included within the scope ofthe present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

What is claimed is:
 1. A method of operating a bioreactor, comprising:performing for a period of time, within the bioreactor, a biochemicalreaction in which at least one eukaryotic cell is grown within a liquidmedium having a volume of less than about 50 milliliters, wherein thebioreactor comprises an online sensor that is in contact with the liquidmedium for monitoring viability of the at least one eukaryotic cell, agas inlet conduit and a gas outlet conduit connected to the bioreactor,and a gas bypass conduit that connects the gas inlet conduit and the gasoutlet conduit, wherein the gas bypass conduit is external to thebioreactor, and wherein, during the period of time, a pressure gradientis applied to an inlet of the bioreactor using a pump and/or to anoutlet of the bioreactor using a syringe; adding a first volume ofliquid to the liquid medium in the bioreactor during the period of time;removing a second volume of liquid from the liquid medium in thebioreactor during the period of time; and monitoring the volume of theliquid within the bioreactor during the period of time; wherein: duringat least about 80% of the period of time, the total volume of liquidwithin the bioreactor does not fluctuate by more than about 20% from anaverage of the volume of liquid within the bioreactor; and over theentire period of time, the osmolarity of liquid within the bioreactor ismaintained within a range of from about 200 osmoles per kilogram of theliquid to about 600 osmoles per kilogram of the liquid.
 2. The method ofclaim 1, wherein the adding step is performed over a first period duringthe period of time, the removing step is performed over a second periodduring the period of time, and the first and second periods do notsubstantially overlap with each other.
 3. A method of operating abioreactor, comprising: performing for a period of time, within thebioreactor, a biochemical reaction in which at least one eukaryotic cellis grown within a liquid medium having a volume of less than about 50milliliters, wherein the bioreactor comprises at least one online sensorthat is in contact with the liquid medium for monitoring viability ofthe at least one eukaryotic cell, a gas inlet conduit and a gas outletconduit connected to the bioreactor, and a gas bypass conduit thatconnects the gas inlet conduit and the gas outlet conduit, wherein thegas bypass conduit is external to the bioreactor, and wherein, duringthe period of time, a pressure gradient is applied to an inlet of thebioreactor using a pump and/or to an outlet of the bioreactor using asyringe; adding a first volume of liquid to the liquid medium in thebioreactor during a first period during the period of time; removing asecond volume of liquid having a volume that is within 10% of the firstvolume of liquid from the liquid medium in the bioreactor during asecond period during the period of time, wherein the first period andthe second period do no overlap; repeating the adding and removing stepsat least one time during the period of time; and monitoring the volumeof liquid within the bioreactor during period of time; wherein: theadding step and the removing step are performed such that, between theadding step and the removing step, substantially no liquid is removedfrom the bioreactor via a nonevaporative pathway, and substantially noliquid is added to the bioreactor during the period of time; and overthe entire period of time, the osmolarity of liquid within thebioreactor is maintained within a range of from about 200 osmoles perkilogram of the liquid to about 600 osmoles per kilogram of the liquid.4. The method of claim 3, wherein the adding step and the removing stepare performed such that, between the adding step and the removing step,substantially no liquid is removed from the bioreactor.
 5. The method ofclaim 3, wherein the second volume of liquid has a volume that is within5% of the volume of the first volume of liquid.
 6. The method of claim1, wherein the biochemical reaction is performed within a liquid mediumhaving a volume of less than about 10 milliliters.
 7. The method ofclaim 1, wherein adding the first volume of liquid to the liquid mediumcomprises transporting the first volume of liquid into the bioreactorvia a liquid inlet.
 8. The method of claim 7, wherein the liquid inletcomprises a channel.
 9. The method of claim 8, wherein the liquid inletchannel comprises a microfluidic channel.
 10. The method of claim 1,wherein removing the second volume of liquid from the liquid medium inthe bioreactor comprises transporting the second volume of liquid out ofthe bioreactor via a liquid outlet.
 11. The method of claim 10, whereinthe liquid outlet comprises a channel.
 12. The method of claim 11,wherein the liquid outlet channel comprises a microfluidic channel. 13.The method of claim 1, wherein the liquid medium comprises a cell growthmedium.
 14. The method of claim 1, wherein at least one eukaryotic cellis grown during the biochemical reaction.
 15. The method of claim 14,wherein the eukaryotic cell is a Chinese hamster ovary (CHO) cell. 16.The method of claim 1, wherein the first volume of liquid added to theliquid medium comprises at least one biochemical reactant.
 17. Themethod of claim 1, wherein the second volume of liquid removed from theliquid medium comprises a product of the biochemical reaction.
 18. Themethod of claim 1, wherein the second volume of liquid removed from theliquid medium comprises a eukaryotic cell.